1. Field of the Invention
The present invention relates generally to microparticles and/or nanoparticles that may be used in biological systems. More particularly, the present invention is directed to modifying the surface chemistry of such particles, without using conventional linking agents, to enhance their compatibility with biological systems and to also provide the particles with one or more biological functions.
2. Description of Related Art
Fluorescent labeling of biological systems is a well-known analytical tool used in modern biotechnology as well as analytical chemistry. Applications for such fluorescent labeling include technologies such as medical (and non-medical) fluorescence microscopy, histology, flow cytometry, fluorescence in-situ hybridization (medical assays and research), DNA sequencing, immunoassays, binding assays, separation, etc.
Conventionally, such fluorescent labeling involves the use of an organic dye molecule bonded to a moiety that, in turn, selectively bonds to a particular biological system, the presence of which is then identified by excitation of the dye molecule to cause it to fluoresce. There are a number of problems with such an analytical system. In the first place, the emission of light of visible wavelengths from an excited dye molecule usually is characterized by the presence of a broad spectrum, i.e., the entire emission spectrum is rather broad. As a result, there is a severe limitation on the number of different color organic dye molecules which may be utilized simultaneously or sequentially in an analysis since it is difficult to either simultaneously or even non-simultaneously detect or discriminate between the presence of a number of different detectable substances due to the broad spectrum emissions and emission tails of the labeling molecules. Another problem is that most dye molecules have a relatively narrow absorption spectrum, thus requiring either multiple excitation beams used either in tandem or sequentially for multiple wavelength probes, or else a broad spectrum excitation source which is sequentially used with different filters for sequential excitation of a series of probes respectively excited at different wavelengths.
Another problem frequently encountered with existing dye molecule labels is that of photostability. Available fluorescent molecules bleach, or irreversibly cease to emit light, under repeated excitation (104-108 cycles of absorption/emission). These problems are often surmounted by minimizing the amount of time that the sample is exposed to light, and by removing oxygen and/or other radical species from the sample. In addition, the probe tools used for the study of systems by electron microscopy techniques are completely different from the probes used for study by fluorescence. Thus, it is not possible to label a material with a single type of probe for both electron microscopy and for fluorescence. This is also the case for multifunctional molecular imaging: Fluorescence+MRI; Fluorescence+PET; Fluorescence+CT; Fluorescence+MRI+PET+CT and any other combination, not even including fluorescence, such as MRI+PET, CT+EM. It would, therefore, be desirable to provide a stable probe material for biological and biomedical applications preferably having a wide absorption band and capable of providing a detectable signal in response to exposure to energy, without the presence of the large red emission tails characteristic of dye molecules (thereby permitting the simultaneous use of a number of such probe materials, each, for example, emitting light of a different narrow wavelength band) and/or capable of scattering or diffracting radiation. It would also be equally desirable to provide a single, stable probe material that can be used to image the same sample by both light and electron microscopy, such as MRI/PET/CT with or without fluorescence.
Semiconductor nanocrystals (NCs or quantum dots) are fragments of semiconductor material composed of a few hundreds to thousands of atoms. Quantum dots have very interesting optical properties resulting from quantum confinement. This confinement occurs when the particles are smaller than the Bohr exciton radius of the material they are composed of.
Since the first synthesis of semiconductor nanoparticles, significant progress has been made to control the size and monodispersion of quantum dots in a range of 1 to 7 nanometers (nm). A special interest was given to quantum dots made from material from the II-IV class such as cadmium and selenide (CdSe). CdSe particles covered with a second layer of zinc/sulfide (CdSe/ZnS) emit a strong fluorescent signal in the visible part of the light spectra. Varying the size of the nanocrystals made of these materials by few nanometers allows the tuning of the emission wavelength while the absorption characteristics are similar for each size.
The chemical synthesis of CdSe/ZnS quantum dots requires a hydrophobic environment and surfactant such as trioctylphosphine oxide (TOPO) in order to control the nucleation between Cd and Se and the growth of the particles. This results in highly hydrophobic particles, poorly soluble in aqueous environments. For biological application using quantum dots as probes, a surface chemistry is thus necessary to remove the surfactant and make the particle biocompatible and soluble in aqueous solvents.
U.S. Pat. Nos. 6,207,392 and 6,423,551 disclose semiconductor nanocrystal probes for biological applications and processes for making and using such probes. The probes include semiconductor nanocrystals, linking agents and affinity molecules. The contents of this patent are hereby incorporated by reference in its entirety.
In U.S. Pat. No. 5,990,479 organo luminescent semiconductor nanocrystal probes for biological applications and process for making and using such probes are disclosed. The contents of this patent are hereby incorporated by reference in its entirety.